The ability to deliver nucleic acids carried by delivery vehicles, e.g., recombinant viruses (adenovirus, adeno-associated virus, herpesvirus, retrovirus); lipid vehicles, poly-lysine vehicles, synthetic polyarnino polymer vehicles which are used with nucleic acid molecules, such as a plasmid, comprising a transgene, to a transfect a target cell; molecular conjugate vectors; and modified viral vectors (adenovirus dodecahedron and recombinant adenovirus conglomerates) to specific cell types is useful for various applications in oncology, developmental biology and gene therapy.
Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb. See generally, Horwitz, M. S., "Adenoviridae and Their Replication," in Virology, 2nd edition, Fields et al., eds., Raven Press, New York, 1990. Recombinant adenoviruses have advantages for use as expression systems for nucleic acid molecules coding for, inter alia, proteins, ribozymes, RNAs, antisense RNA that are foreign to the adenovirus carrier (i.e. a transgene), including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts. See Berkner, K. L., 1992, Curr. Top. Micro Immunol, 158:39-66; Jolly D., 1994, Cancer Gene Therapy, 1:51-64.
Adenoviruses have a natural tropism for respiratory tract cells, which has made them attractive vectors for use in delivery of genes to respiratory tract cells. For example, adenovirus vectors have been and are being designed for use in the treatment of certain diseases, such as cystic fibrosis (CF): the most common autosomal recessive disease in Caucasians. In CF, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disturb cAMP-regulated chloride channel function, resulting in pulmonary dysfunction. The gene mutations have been found to encode altered CFTR proteins which cannot be translocated to the cell membrane for proper functioning. The CFTR gene has been introduced into adenovirus vectors to treat CF in several animal models and human patients. Particularly, studies have shown that adenovirus vectors are fully capable of delivering CFTR to airway epithelia of CF patients, as well as airway epithelia of cotton rats and primates. See e.g., Zabner et al., 1994, Nature Genetics, 6:75-83; Rich et al., 1993, Human Gene Therapy, 4:461-476; Zabner et al., 1993, Cell, 75:207-216; Zabner et al., 1994, Nature Genetics 6:75-83; Crystal et al., 1004, Nature Genetics, 8:42-51; Rich et al., 1993, Human Gene Therapy, 4:461-476.
However, it would be useful to alter the tropism of a virus, such as adenovirus, to allow it to be used to deliver a nucleic acid molecule to a variety of cells for which the virus is normally non-tropic.
Adenoviruses are about 65-80 nm in diameter and are regular icosahedrons, having 20 triangular surfaces and 12 vertices. A "fiber" projects from each vertex. There are currently approximately 42 known serotypes of adenovirus. The individual serotypes have different properties such as different fiber lengths. The protein coat, or capsid, of the adenovirus has approximately 252 subunits: 240 "hexons" and 12 "pentons". The pentons each have a penton base on the surface of the capsid and a fiber which projects from the base. Each fiber is surrounded by 5 hexons. The hexons and pentons are derived from 25 different viral polypeptides. Horwitz, M. S., "Adenoviridae and Their Replication", in Virology, 2nd ed., Fields et al., eds., Raven Press, New York, 1990, p. 1680.
As presently understood, adenovirus enters cells, e.g., in the respiratory tract, by attaching via the fiber to a cell surface receptor (known as CAR for Coxsackie adenovirus receptor) on the cell membrane of the host cell. The virus attached to its receptor migrates into the cell, within the plasma membrane to clathrin-coated pits, which form endocytic vesicles or receptosomes. Horwitz, M. S., "Adenoviridae and Their Replication", in Virology, 2nd ed., Fields et al., eds., Raven Press, New York, 1990, p. 1680. When the virus reaches the nuclear pores, the viral DNA enters the nucleus, leaving many virion proteins in the cytoplasm. Horwitz, M. S., "Adenoviridae and Their Replication", in Virology, 2nd ed., Fields et al., eds., Raven Press, New York, 1990, p. 1680.
It would be useful to mediate infection of the host cell by controlling the targeting of the adenovirus to cell surface molecules to which adenovirus does not normally bind. In this way the rate of infection can be controlled and the adenovirus can be targeted to certain cells or tissues within an organism.
Like adenoviruses, retroviruses have also been used for delivery of transgenes to target cells. As set forth above, a transgene is a nucleic acid molecule that codes for, inter alia, a protein, RNA, ribozyme, antisense RNA not produced by the virus. Retrovirus virions range in diameter from 80 to 130 nm and are made up of a protein capsid that is lipid encapsulated. The viral genome is encased within the capsid along with the proteins integrase and reverse transcriptase. The retrovirus genome consists of two RNA strands. After the virus enters the cells, the reverse transcriptase synthesizes viral DNA using the viral RNA as its template. The cellular machinery then synthesizes the complementary DNA which is then circularized and inserted into the host genome. Following insertion, the viral RNA genome is transcribed and viral replication is completed.
Examples of retroviruses include Moloney murine leukemia virus (Mo-MuLV), HTLV and HIV retroviruses. Mo-MuLV vectors are most commonly used and are produced simply by replacing viral genes required for replication with the desired transgenes to be transferred. The genome in retroviral vectors contains a long terminal repeat sequence (LTR) at each end with the desired transgene or transgenes in between. The most commonly used system for generating retroviral vectors consists of two parts, the retroviral vector and the packaging cell line.
Retroviruses are typically classified by their host range. For example, ecotropic viruses are viruses which bind receptors unique to mice and are only able to replicate within the murine species. Xenotropic viruses bind receptors found on all cells in most species except those of mice. Polytropic and amphotropic viruses bind different receptors found in both murine and nonmurine species. The host range is determined primarily by the binding interaction between viral envelope glycoproteins and specific proteins on the host cell surface that act as viral receptors. For example, in murine cells, an amino acid transporter serves as the receptor for the envelope glycoprotein gp70 of ecotropic Moloney murine leukemia virus (Mo-MuLV). The receptor for the amphotropic MoMuLV has recently been cloned and shows homology to a phosphate transporter. There are six known receptors for retroviruses: CD4 (for HIV); CAT (for MLV-E (ecotropic Murine leukemic virus E); RAMl/GLVR2 (for murine leukemic virus-A (MLV-A)); GLVRI (for Gibbon Ape leukemia virus (GALV) and Feline leukemia virus B (FeLV-B). RAM1 and GLVR1 receptors are broadly expressed in human tissues.
Retrovirus packaging cell lines provide all the viral proteins required for capsid production and the virion maturation of the vector, i.e., the gag, pol and env genes. For the MMLV vectors, it is the packaging cell line that determines whether the vector is ecotropic, xenotropic or amphotropic. The choice of the packaging cell line determines the cells that will be targeted. Thus, the usefulness of retroviruses for gene transfer is limited by the fact that they are receptor specific.
However, retroviruses are useful for gene delivery systems because they have a high infection efficiency and the retroviral nucleic acid (after reverse transcription) integrates into the host genome resulting in sustained expression of the transgenes carried by the vector. However, typical retroviral vectors are limited in that they require dividing cells for infectivity. Furthermore, in vivo delivery of these vectors is poor and is effective only when infecting helper cell lines. Thus, it would be useful to have a system for increasing the efficiency of retroviral infection.
Certain situations exist where it would be useful to modify the tropism of viruses to target the vector to cell surface molecules other than the virus' normal cells surface receptor. For example, certain cells are normally refractory to infection by certain viruses. It would be useful to have a method of overcoming the inability of these cells to be infected. Similarly, in cancer cells, many receptors are up-regulated. Therefore, it would be useful to be able to specifically target vectors to these up-regulated receptors to increase the uptake of nucleic acids providing for antitumor agents for treatment of such cancers. For example, in Karposi's sarcoma (Prism), there is an increase in the number and activity of receptors for fibroblast growth factor. Thus, this receptor would function as a useful target for infection/transfection. There are a number of additional cell surface molecules that are also specific for various cell types and it would be useful to employ these molecules for targeted infection/transfection as well.
In cystic fibrosis (CF) studies, airway epithelial cells have been infected with adenovirus comprising DNA encoding for CFTR. However, the efficiency of infection of such vectors has proven to be low, leading to a low efficiency of CFTR DNA expression in cells. Furthermore, in practice, in order to achieve effective transgene transfer into affected cells, the viral vector comprising the transgene is repeatedly administered over a course of time. Such readministration of the viral vector can trigger an immune response within the subject to whom the vector is given, which requires subsequently higher doses or an elimination of infection. If the efficiency of uptake of virus is increased, a lower dose of virus can be used to alleviate certain conditions, which, in turn, may help alleviate the immune response problems which are associated with the readministration of vectors. Thus, it would be useful to have a mechanism by which the targeting of the viral vector can be controlled.
Other delivery vehicles comprising cationic amphiphiles such as lipids, synthetic polyamino polymers (Goldman et al., 1997, Nat. Biotechnol. 15:462-466), and poly-lysine (Kollen et al., 1996, Hum. Gene. Ther. 7:1577-1586), vehicles which are used with nucleic acid molecules, such as a plasmid, comprising a transgene, to transfect a target cell, are useful for delivery of nucleic acids to cells (herein after described as lipid vehicle, synthetic polyamino polymer vehicles, and poly-lysine vehicles). Most of these delivery vehicles suffer from nonspecificity and inefficiency of delivery. Therefore, a method for targeting these systems to cells would also be useful.
Additionally, modified viral systems utilizing the adenovirus dodecahedron which consists of a dodecahedron made of adenovirus pentons or penton bases which are a complex of a penton base and a fiber and allow for internalization and liberation of virus into the cytoplasm (Fender et al., 1997, Nature Biotechnology 15:52-56), and recombinant adenovirus conglomerates where the transgene to be transferred is in the genome of a recombinant replication-incompetent adenovirus which also acts as the endosomolytic agent (Schwarzenberger et al., 1997, J. Virol; 71:8563-8571) are useful for the delivery of nucleic acids to cells. These delivery vehicles suffer the same inadequacies as discussed above for virus delivery vehicles (e.g. low infectivity). A system which allows for specific targeting of these delivery vehicles would be advantageous.
U.S. Pat. No. 5,574,146 ('146) discloses a method for targeted oligonucleotide delivery to cells utilizing proteolytically cleavable peptides to link an oligodeoxynucleotide (ODN) to a targeting moiety. The '146 invention is dependent upon the proteolytically cleavable peptide being cleaved upon entry into the cell by lysosomal proteases, thereupon releasing the ODN within the cell. Furthermore, the invention in the '146 patent requires the use of automated techniques to synthesize the ODN sequences in order to produce an ODN whose nucleotide sequence comprises a reactive group capable of linking to a reactive group of the proteolytically cleavable peptide. This synthesis requirement limits the length of the ODN in the range of 5 to 100 nucleotides due to the limits of automated synthesis. Such short ODNs generally would not be sufficient to encode a full-length protein or polypeptide molecule capable of replacing an endogenous mutated molecule or providing for an otherwise desirable non-existent full-length protein, RNA, DNA, antisense molecule in the cell. Moreover, the '146 patent necessitates the use of a lysosomotropic agent for the internalization of the ODN into the cell. Therefore, the invention disclosed in the '146 patent is significantly limited in scope to the delivery of small nucleic acid molecules (5 to 100 nucleotides in length) through the use of proteolytically cleavable peptides via lysosomotropic agents.
Additionally, WO 98/39464, WO 98/39465, and WO 98/39467 disclose a method for targeting specific cell populations to express a protein of interest. This is achieved by the use of a recombinant adenovirus vector, which comprises a first adenovirus gene under transcriptional control of a first heterologous transcriptional regulatory element (TRE), and at least a second gene under control of a second heterologous TRE, wherein the first heterologous TRE is cell-specific, and both heterologous TREs are functional in the same cell. The first adenovirus gene is essential for adenovirus replication and the second gene may be a transgene of interest. The recombinant adenovirus vectors are thereby limited to replicating only in the target cell, although all cells may be infected with the recombinant adenovirus vector. The invention also provides for a method whereby the immunogenicity of the recombinant adenovirus vector is masked by being complexed with35 a non-immunogenic hydrophillc polymer which may be complexed to the recombinant adenovirus vector by covalent or non-covalent attachment to the capsid proteins of the virus. The preferred hydrophilic polymer is polyethylene glycol covalently linked through a tresyl-MPEG (TMPEG) to adenovirus ("linked through .epsilon.-amino groups on lysine residues on the adenovirus using TMPEG). Such treatment of the virus is not involved in the targeting mechanism and solely achieves the task of reducing the immunogenicity of the recombinant virus vector. The three aforementioned patent applications all employ a similar method although the TREs vary among them. The potential problem with such an invention is that it cannot ensure that cells that are not being targeted but may be infected will not exhibit any leaky expression of the transgene. Where the transgene of interest is a cytotoxic gene, leaky expression would be highly undesirable.
It would be useful to have a method of linking nucleic acid delivery systems capable of delivering a nucleic acid molecule, regardless of composition and size, to specific cell surface molecules to stimulate uptake of the delivery vehicle into the cells such that only the cells that are targeted will internalize the delivery vehicle.
For example, major histocompatibility complex ("MHC") molecules are found in on essentially all nucleated cells and assist in T-cell mediated immune response. In human cells, MHC molecules are also called human-leukocyte antigens ("HLA antigens"), There are two classes of MHC molecules, class I and class II. Class I MHC molecules are polymorphic integral membrane proteins that bind a diverse group of foreign antigens or self-antigens for presentation to T cells. The extracellular portion of Class I MHC heavy (H) chains comprises three structural domains, .alpha..sub.1, .alpha..sub.2 and .alpha..sub.3. These H chains are noncovalently associated in an equimolar ratio with .beta..sub.2 -microglobulin, a soluble, nonpolymorphic protein. Helper T cells react against foreign Class II glycoproteins. The Class II glycoproteins are composed of two noncovalently bonded polypeptide chains: an .alpha.-chain with a molecular weight of about 33,000 and a .beta.-chain with a molecular weight of about 28,000. MHC molecules are found on a wide variety of cell types and are efficiently internalized by endocytosis in numerous cell types.
Alberts, et al. indicates that antigens seen by T cells are degraded inside a host cell before they are presented to the T cell on the surface of the host sell. The fragments of viral proteins wind up on the surface of the infected cell by associating with MHC molecules either on the surface of the cells or perhaps inside the cell. Alberts, et al., 1986, Molecular Biology of the Cell, 2.sup.nd ed., p. 1043.
Class I MHC molecules are continuously shuttling peptides back and forth from the endoplasmic reticulum (ER) to the plasma membrane at the surface of the cell. The MHC peptide complex can bind to the T-cell receptor complex which in turn leads to activation of the T-cell. The structure and the fate of Class I MHC molecules in both the ER and on the cell surface are regulated by peptides in the cell and .beta..sub.2 -microglobulin. For example, a high local concentration of .beta..sub.2 -microglobulin plays an important role in maintaining Class I MHC chains in a conformation accessible to peptides. This high level of .beta..sub.2 -microglobulin in the ER can increase the efficiency with which Class I MHC molecules bind peptides for transport to the cell surface. Conversely, the inherent instability of free Class I MHC H chains in the presence of low .beta..sub.2 -microglobulin concentrations serves to limit the number of cell surface class I molecules that can capture extracellularly derived antigenic peptides for presentation to T-cells.
Roux et al. described an approach to target nucleic acid molecules to specific cell types using retroviruses. Roux, P., et al., 1989, Proc. Nat. Acad. Sci. USA, 86, 9079-9083. This approach used biotinylated antibodies to the retroviral envelope protein connected to biotinylated antibodies to specific cell membrane markers by streptavidin. According to this method, a first bifunctional antibody complex containing a biotinylated anti-major histocompatibility complex (MHC) antibody is added to the cells which are to be infecteditransfected. The cells are then washed and incubated with streptavidin, and then washed again. This process results in a cell/anti-MHC-biotin/streptavidin complex. Then the retrovirus of interest is incubated with biotinylated anti-gp-70 antibodies to create precoated retroviruses. Gp-70 is a virus-encoded glycoprotein which binds to specific cell membrane receptors. The precoated retroviruses are then added to the MHC-streptavidin complex. The result is a linking of the retrovirus to the MHC cell receptor via a biotinylated anti-MHC antibody/streptavidinlbiotinylated anti-gp-70 antibody bridge. As stated by the authors, a major limitation of this approach is the relatively low infection yield. Roux, P., et al., 1989, Proc. Nat. Acad. Sci. USA, 86, 9079-9083. Thus, the usefulness of this approach is rather limited. Furthermore, this approach cannot be used in vivo because the target cells in vivo cannot be pre-incubated with streptavidin. Furthermore, streptavidin is known to be immunogenic. Marshall, D., et al., 1996, British Journal of Cancer, 73 (5):562-72.
Other examples of useful specific cell surface receptors include the following: (1) The folate receptor. The folate receptor is over-expressed on the cell surface of a variety of human tumors, including those of the ovary, kidney, uterus, testis, brain, colon, lung and myelocytic blood cells, and can be used as a receptor for targeted delivery vehicles. Either folate, or an antibody against the folate receptor (Melani et al., 1998, Cancer Res. 58(18):4146-4154) can be bound to the receptor for use as cell surface binding portions. (2) The transferrin receptor. The expression of the transferrin receptor correlates with cellular proliferation, its levels being highest among dividing cells, including a variety of tumor cells. Shindelman et al., 1981, Int J Cancer 27:329-334. In breast tissue, transferrin receptor expression has been related to the presence of malignancy (Faulk et al., 1980, Lancet 2:390-392). Transferrin receptor expression also occurs in lung and colon adenocarcinomas, some sarcomas and some forms of Hodgkin's disease. Gatter et al., 1983, J. Clin. Pathol. 36:539-545. Transferrin, or an antibody against the transfernin receptor, for example HB21, a Fab fragment of a monoclonal antibody directed against the human transferrin receptor (Debinski and Pastan, 1992, Cancer Res. 52:5379-5385), can be used for targeting the transferrin receptor. (3) The fibroblast growth factor (FGF) receptor. FGF receptors are endogenously expressed on the cell surface of Karposi sarcoma cells. Goldman et al., 1997, Cancer Res. 57:1447-1451. Karposi sarcoma is a major AIDS related malignancy associated with a significant morbidity and mortality.
Fibroblast growth factor (FGF2) can be used to target cells expressing the FGF receptor. (4) Epidermal growth factor (EGF) receptor. As with the FGF receptor, the EGF receptor may be targeted by the epidermal growth factor (EGF). Bell et al., 1986, Nucleic Acids Res. 21:8427-8447. (5) The c-kit receptor may also be targeted by molecules that bind to it. Schwarzenberger et al., 1996, Blood 87:472-478. (6) The erythrocyte growth factor receptor is also up-regulated on the cell surface of many tumor cells. It can be targeted using an antibody to the receptor, e.g. monoclonal antibody B4G7. Shimizu et al., 1996, Cancer Gene Therapy 3:113-120. The receptor may also be targeted using the erythrocyte growth factor. (7) Polymeric Ig Receptor. The polymeric Ig receptor is expressed on the basal cell surface of respiratory epithelial cells and are therefore particularly advantageous for targeting since they represent an alternative mechanism for entry into the cell via the bloodstream which is potentially more amenable for therapeutics. Fab fragments against the polymeric Ig receptor may be used for targeting respiratory epithelial cells. (8) The erythropoietin (EPO) receptor can also be used for targeting cells via erythropoietin. Yoshimura and Misawa, 1998, Curr Opin Hematol. 5:171-176. (9) The purinoceptor. O'Reilly et al., 1998, Br. J. Pharmacol. 124:1597-1606. The purinoceptor can be targeted with purine or purine analogs (e.g. ATP, UTP, ATP-.gamma.-S, AMP-PNP, and INS 365) which bind to the purinoceptor. INS 365 is a purinoceptor agonist described by Shaffer et al. Shaffer et al., 1998, Pediatric Pulmonology, Supplement 17, Abstract 198.
Additionally, certain enzymes are found on the cell surface (e.g. metalloproteases) which could also serve as targets of delivery vehicles via small molecules, peptides, or antibodies that bind to these cell surface molecules
Small molecules may be particularly useful in targeting delivery vehicles to cells as they may circumvent proteolysis problems that may interfere with the usefulness of peptides, proteins, and antibodies as targeting molecules.